Pichia pastoris, a methylotrophic yeast, is known to be an efficient host for heterologous proteins production. In this study, a recombinant P. pastoris Y11430 was found better for β-glucosidase activity in comparison with a wild type P. pastoris Y11430 strain, and thereby, subjected to methanol intermittent feed profiling for β-glucosidase production. The results showed that at 72 h of cultivation time, the cultures with 16.67% and 33.33% methanol feeding with constant rate could produce the total dry cell weight of 52.23 and 118.55 g/L, respectively, while the total mutant β-glucosidase activities were 1001.59 and 3259.82 units, respectively. The methanol feeding profile was kept at 33% with three methanol feeding strategies such as constant feed rate, linear feed rate, and exponential feed rate which were used in fed-batch fermentation. At 60 h of cultivation, the highest total mutant β-glucosidase activity was 2971.85 units for exponential feed rate culture. On the other hand, total mutant β-glucosidase activity of the constant feed rate culture and linear feed rate culture were 1682.25 and 1975.43 units, respectively. The kinetic parameters of exponential feed rate culture were specific growth rate on glycerol 0.228/h, specific growth of methanol 0.061/h, maximum total dry cell weight 196.73 g, yield coefficient biomass per methanol ([Formula: see text]) 0.57 gcell/gMeOH, methanol consumption rate ([Formula: see text]) 5.76 gMeOH/h, and enzyme productivity ([Formula: see text]) 75.96 units/h. In conclusion, higher cell mass and β- glucosidase activity were produced under exponential feed rate than constant and linear feed rates.
The consequence of the massive increase in population in recent years is the enormous production of mainly industrial waste. The effort to minimize these waste products is, therefore, no longer sufficient. Biotechnologists, therefore, started looking for ways to not only reuse these waste products, but also to valorise them. This work focuses on the biotechnological use and processing of waste oils/fats and waste glycerol by carotenogenic yeasts of the genus Rhodotorula and Sporidiobolus. The results of this work show that the selected yeast strains are able to process waste glycerol as well as some oils and fats in a circular economy model and, moreover, are resistant to potential antimicrobial compounds present in the medium. The best-growing strains, Rhodotorula toruloides CCY 062-002-004 and Rhodotorula kratochvilovae CCY 020-002-026, were selected for fed-batch cultivation in a laboratory bioreactor in a medium containing a mixture of coffee oil and waste glycerol. The results show that both strains were able to produce more than 18 g of biomass per litre of media with a high content of carotenoids (10.757 ± 1.007 mg/g of CDW in R. kratochvilovae and 10.514 ± 1.520 mg/g of CDW in R. toruloides, respectively). The overall results prove that combining different waste substrates is a promising option for producing yeast biomass enriched with carotenoids, lipids, and beta-glucans.
- Publication type
- Journal Article MeSH
OBJECTIVE: Desalination of cheese whey by electrodialysis yields saline wastewater (SWW). The goal was to test this as the basis of a culture medium and to prove experimentally the concept that it was a suitable resource for heterotrophic cultivation of the freshwater green microalga Chlorella vulgaris. RESULTS: Optimization of glucose concentration, nitrogen source and medium salinity for microalgal growth was first carried out in defined medium (DM) and shake flasks. These results were then adopted in shake flask cultivation experiments using pre-treated SWW medium (PSWW). Subsequently, microalgal growth under optimized conditions was tested in bioreactors. Various media such as DM, PSWW and diluted PSWW (DPSWW) were compared. Volumetric biomass productivities decreased in the order DM (0.371 g L-1 h-1, urea) > DPSWW (0.315 g L-1 h-1, soy peptone) > PSWW (0.152 g L-1 h-1, soy peptone). Although biomass productivities in DPSWW and PSWW media were significantly lower than in DM, these media required the addition of only 66 and 33% of DM N sources, respectively. No other added DM component was necessary in (D)PSWW to achieve microalgal growth. CONCLUSIONS: Although the optimized cultivation of freshwater microalgae on alternative medium based on SWW resulted in biomass productivities lower than those on DM, the required addition of N sources was also lower. Potentially lower production costs of Chlorella biomass and the meaningful use of SWW are the main outcomes of this work.
Two experiments were carried out to examine the impacts of hydroxytyrosol (HT) on lipid metabolism and mitochondrial function in Megalobrama amblycephala. Triplicate groups of fish were fed four test diets: (1) low-fat diet (LFD, 5% fat), (2) high-fat diet (HFD, 15% fat), (3) LFD + 100 mg/kg HT (LFD + HT), and (4) HFD + 100 mg/kg HT (HFD + HT) (in vivo). Hepatocytes from the same batch were exposed to three media including L-15 medium (L15), oleic acid (OA) medium [L15 + 400 μM OA], and OA + HT medium [L15 + 400 μM OA + 10 μM HT] to explore the roles of HT in mitochondrial function (in vitro). Fish fed HFD had excessive fat deposition in the liver, and HT inclusion in the HFD decreased hepatic fat deposition. Transmission electron microscopy revealed that the HFD triggers loss of cristae and metrical density and hydropic changes in mitochondria and that HT supplementation attenuates the ultrastructural alterations of mitochondria. The in vitro test showed that HT decreases fat deposition in hepatocytes, suppresses the reactive oxygen species formation, and facilitates the expression of phospho-AMPK protein and the genes involved in mitochondria biogenesis (PGC-1, NRF-1, TFAM) and autophagy (PINK1, Mul1, Atg5). These findings suggest the lipid-lowering effect of HT mediated by activation of mitochondrial biogenesis and autophagy through the AMPK pathway.
- MeSH
- Autophagy * MeSH
- Organelle Biogenesis MeSH
- Cyprinidae genetics metabolism MeSH
- Dietary Fats metabolism MeSH
- Phenylethyl Alcohol analogs & derivatives metabolism MeSH
- Hepatocytes metabolism MeSH
- Liver cytology metabolism MeSH
- Animal Feed analysis MeSH
- Mitochondria metabolism MeSH
- AMP-Activated Protein Kinases genetics metabolism MeSH
- Fish Proteins genetics metabolism MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
The aim of the study was to identify the optimum cultivation conditions for the microalgal growth and lipid production of the oleaginous microalga Chlorella pyrenoidosa Chick (IPPAS C2). Moreover, an appropriate NO3- concentration in the cultivation medium for maximized lipid accumulation was determined. The experimental design involved a biphasic cultivation strategy with an initial biomass accumulating phase under optimized light (400 μmol/m2 per s), temperature (25 °C), and elevated CO2 concentration in the air mixture (3%), followed by a mid-elevated CO2 concentration (0.5%) for lipid induction. The highest lipid yields of 172.47 ± 18.1 and 179.65 ± 25.4 mg/L per day were detected for NO3- concentrations of 100 and 150 mg/L. The optimization approach presented here led not only to the maximization of lipid yield but also to the development of a biphasic cultivation strategy easily applicable to the cultivation process without the necessity for algal cell harvesting between the first and second cultivation phases.
- MeSH
- Biomass MeSH
- Chlorella growth & development metabolism MeSH
- Nitrates metabolism MeSH
- Photobioreactors MeSH
- Culture Media metabolism MeSH
- Lipids biosynthesis MeSH
- Microalgae growth & development metabolism MeSH
- Carbon Dioxide metabolism MeSH
- Batch Cell Culture Techniques MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
Pathogenic yeasts Candida albicans and Candida parapsilosis possess a ß-type carbonic anhydrase Nce103p, which is involved in CO2 hydration and signaling. C. albicans lacking Nce103p cannot survive in low CO2 concentrations, e.g., in atmospheric growth conditions. Candida carbonic anhydrases are orthologous to the Saccharomyces cerevisiae enzyme, which had originally been detected as a substrate of a non-classical export pathway. However, experimental evidence on localization of C. albicans and C. parapsilosis carbonic anhydrases has not been reported to date. Immunogold labeling and electron microscopy used in the present study showed that carbonic anhydrases are localized in the cell wall and plasmatic membrane of both Candida species. This localization was confirmed by Western blot and mass spectrometry analyses of isolated cell wall and plasma membrane fractions. Further analysis of C. albicans and C. parapsilosis subcellular fractions revealed presence of carbonic anhydrases also in the cytosolic and mitochondrial fractions of Candida cells cultivated in shaken liquid cultures, under the atmospheric conditions.
- MeSH
- Cell Membrane enzymology MeSH
- Cell Wall enzymology MeSH
- Candida albicans enzymology growth & development MeSH
- Candida parapsilosis enzymology growth & development MeSH
- Cytosol enzymology MeSH
- Microscopy, Electron MeSH
- Fungal Proteins metabolism MeSH
- Mass Spectrometry MeSH
- Carbonic Anhydrases metabolism MeSH
- Mitochondria enzymology MeSH
- Batch Cell Culture Techniques MeSH
- Publication type
- Journal Article MeSH
The aim of the study was to screen Yarrowia lipolytica strains for keto acid production and determine optimal conditions for pyruvic acid biosynthesis from glycerol by the best producer. The analyzed parameters were thiamine concentration, medium pH, stirring speed, and substrate concentration. The screening was performed in flask cultures, whereas pyruvic acid production was carried out in 5-L stirred-tank reactor with 2 L of working volume. In total, 24 Y. lipolytica strains were compared for their abilities to produce pyruvic and α-ketoglutaric acids. The total concentration of both acids ranged from 0.1 to 15.03 g/L. Ten strains were selected for keto acid biosynthesis in bioreactor. The Y. lipolytica SKO 6 strain was identified as the best producer of pyruvic acid. In the selected conditions (thiamine concentration 1.5 μg/L, pH 4.0, stirring speed 800 rpm, 150 g/L of glycerol), the strain Y. lipolytica SKO 6 produced 99.3 g/L of pyruvic acid, with process yield of 0.63 g/g and volumetric production rate of 1.18 g/L/h. Higher titer of pyruvic acid was obtained during fed-batch culture with 200 g/L of glycerol, reaching 125.8 g/L from pure glycerol (yield 0.68 g/g) and 124.4 g/L from crude glycerol (yield 0.62 g/g). Results obtained for the strain Y. lipolytica SKO 6 proved the suitability of microbial production of pyruvic acid at industrial scale.
- MeSH
- Bioreactors MeSH
- Glycerol analysis metabolism MeSH
- Culture Media chemistry MeSH
- Pyruvic Acid analysis metabolism MeSH
- Ketoglutaric Acids analysis metabolism MeSH
- Batch Cell Culture Techniques MeSH
- Thiamine analysis MeSH
- Yarrowia growth & development metabolism MeSH
- Publication type
- Journal Article MeSH
Grass silage as a renewable feedstock for an integrated biorefinery includes nutrients and carbon sources directly available in the press juice (PJ) and in lignocellulosic saccharides from the plant framework. Here, a novel two-stage fed-batch fermentation process for biosynthesis of poly-3-hydroxybutyrate (PHB) by Cupriavidus necator DSM 531 is presented. For bacterial growth, nutrient-rich PJ was employed as a fermentation medium, without any supplements. Saccharides derived from the mechano-enzymatic hydrolysis of the press cake (PC) were subjected to a lactic acid fermentation process, before the fermentation products were fed into the polymer accumulation phase. By combination of pH-stat feeding and cell recycling, the PHB content in 22 g L-1 total-dry cells reached 39% after 32 h of cultivation. Using mimicked hydrolyzate of diluted PJ artificially supplemented with glucose and xylose, the resulting cell dry weight of 21 g L-1 contained 42% PHB.
- MeSH
- Bioreactors MeSH
- Cupriavidus necator MeSH
- Fermentation * MeSH
- Hydroxybutyrates metabolism MeSH
- Poaceae * MeSH
- Polyesters metabolism MeSH
- Silage MeSH
- Publication type
- Journal Article MeSH
The pharmaceutical production of recombinant proteins, such as monoclonal antibodies, is rather complex and requires proper development work. Accordingly, it is essential to develop appropriate scale-down models, which can mimic the corresponding production scale. In this work, we investigated the impact of the bioreactor scale on intracellular micro-heterogeneities of a CHO cell line producing monoclonal antibodies in fed-batch mode, using a 10 mL micro-bioreactor (ambr™) scale-down model and the corresponding 300 L pilot-scale bioreactor. For each scale, we measured the time evolution of the proteome, which enabled us to compare the impact of the bioreactor scale on the intracellular processes. Nearly absolute accordance between the scales was verified by data mining methods, such as hierarchical clustering and in-detail analysis on a single protein base. The time response of principal enzymes related to N-glycosylation was discussed, emphasizing major dissimilarities between the glycan fractions adorning the heavy chain and the corresponding protein abundance. The enzyme expression displayed mainly a constant profile, whereas the resulting glycan pattern changed over time. It is concluded that the enzymatic activity is influenced by the changing environmental conditions present in the fed-batch processes leading to the observed time-dependent variation.
- MeSH
- Models, Biological * MeSH
- Bioreactors * MeSH
- CHO Cells MeSH
- Cricetulus MeSH
- Glycosylation MeSH
- Cricetinae MeSH
- Antibodies, Monoclonal metabolism MeSH
- Cell Proliferation MeSH
- Proteomics methods MeSH
- Recombinant Proteins metabolism MeSH
- Cluster Analysis MeSH
- Animals MeSH
- Check Tag
- Cricetinae MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Hlavním cílem tohoto projektu bylo navrhnout a optimalizovat proces mikrobiální produkce kyseliny mléčné na modelovém MRS médiu za použití levného keratinového hydrolyzátu s cílem dosáhnout vysokého výtěžku a koncentrace kyseliny mléčné, produkované kmenem Lactobacillus casei 198. Nejprve bylo kuřecí peří zkoumáno jako levný zdroj dusíku. S ohledem na růst biomasy a konečnou koncentraci kyseliny mléčné, jako hlavního kritéria, byl pro další experimenty vybrán 20 % hydrolyzát kuřecího peří. Použití tohoto hydrolyzátu způsobilo minimální zředění celkového objemu kultivačního média v bioreaktoru. Konečná dosažená koncentrace kyseliny mléčné v médiu ze vsádkové kultivace byla 48 g/l. Bohužel, i když hydrolyzát peří vykazoval slibný výsledek jako neutralizační činidlo ve vsádkové kulti‑ vaci, tak se neprokázal jako vhodný zdroj dusíku v přítokované kultivaci, kde bylo dosaženo pouze 35 % koncentrace kyseliny mléčné ve srovnání s MRS médiem.
The main objective of this project was to design and optimize the process of microbial production of lactic acid on a model MRS medium using an inexpensive keratin hydrolysate with the goal to achieve high yield and concentration of lactic acid produced by the strain Lactobacillus casei 198. Firstly, the chicken feather was investigated as a low‑cost source of nitrogen. Taking the growth of biomass and the final concentration of lactic acid as the main criteria, 20 % hydrolysate of chicken feather was chosen for the next experiments. Using this hydrolysate caused minimal dilution of the total volume of the culture medium in the bioreactor. The final achieved concentration of lactic acid in the medium in batch culture was 48 g/L Unfortunately, although the feather hydrolysate showed promising result as a neutralizing agent in a batch culture, it was not suitable as a source of nitrogen in a fed‑batch culture, where only 35 % concentration of lactic acid was achieved compared to the MRS medium.
- MeSH
- Chemical Engineering methods MeSH
- Lactic Acid * biosynthesis economics MeSH
- Waste Management MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
